Immunoassay method makes use of antigen-antibody reaction, and is used for detection or quantitative determination of various substrates. This immunoassay method is broadly divided into competitive reaction method, and anti-competitive reaction method. Each method is divided into heterogeneous method which BF-separates antigen-antibody reaction bound form (B: Bound Form) from non-bound free form (F: Free Form), and homogeneous method which does not involve BF separation. The heterogeneous method includes liquid phase method where antigen-antibody reaction proceeds in liquid phase, and solid phase method where it proceeds in the interface between solid phase and liquid phase. For the determination of antigen or antibody, anti-competitive reaction method, in particular heterogeneous-solid phase method such as sandwich method, would apply more often than competitive reaction method since the former shows excellent detection sensitivity.
Exemplary procedures for determining an antigen by sandwich method is as follows: an antibody against an antigen to be determined is bound to a solid-phase support, and thereto is added a test liquid containing the antigen to be determined, to thereby initiate the antigen-antibody reaction (first reaction) in which the antigen specifically binds to the antibody, followed by washing for elimination of contaminants. Then, a labeled substance, for example enzyme-bound enzymatic labeled antibody, is added for initiation of antigen-antibody reaction (second reaction) to thereby bind the enzymatic labeled antibody to the antigen which is bound to the solid-phase support. Subsequently, it is washed for BF separation. For the measurement of enzymatic activity of the enzymatic labeled antibody that was bound to the antigen, a reagent containing a colorimetric substrate or a luminescent substrate is added for initiating enzymatic reaction, and absorbance or luminescence is determined to assay the amount of the antigen.
An example of determination of an antigen by competitive heterogeneous method is as follows: an antibody against an antigen to be determined is bound to a solid-phase support, and thereto are added simultaneously a test liquid containing the antigen to be determined, and a labeled antigen in which a labeled substrate is bound to said antigen, to thereby initiate antigen-antibody reaction for competitive reaction with the antibody in the solid-phase support. In this antigen-antibody reaction, the antigen to be determined and the labeled antigen are bound to the solid-phase support, according to a ratio of respective amount. As in the case of sandwich method, BF separation is then performed to initiate enzymatic reaction. The enzymatic activity in this case depends on an amount of the labeled antigen that was bound, so the amount of the antigen to be determined is determined using calibration curve on the basis of the amount of the labeled antigen added.
Patent document 1 and Patent document 2 describe reaction vessels for use in all of these enzymatic immunoassays. The reaction vessel disclosed in Patent document 1 adopts a structure in which permeable division walls are provided at the upper and lower edges of capillary narrow tubes, and microparticles are packed between these division walls. With binding an antigen or antibody to the microparticles, the narrow tubes are filled with a test liquid to initiate the antigen-antibody reaction. The same applies to the reaction vessel disclosed in Patent document 2 which adopts a structure where microparticles bound to an antigen or antibody are packed in the narrow tubes. Further, in the case of Patent document 2, microparticles are immersed and kept in a preservative solution, while closed-end vent communicates with the narrow tubes at its downstream. The vent is opened upon the assay to thereby enable the passage of the preservative solution and test solution.
Patent document 1: JP Sho62-169,055, A
Patent document 2: JP 2,515,392, B